Analysis of analytes in various biological fluids is of great interest for the diagnosis of diseases and disorders. Generally, the presence of particulates and cellular matter in the fluids interfere with the assay procedures. For example, when analyzing for analytes in blood, the removal of Red Blood Cells (RBC) is often necessary to get meaningful results.
Conventional methods such as centrifugation, sedimentation, etc can be readily used to separate RBCs. However, the conventional methods suffer from the disadvantage that they require separate equipment and technician time for separation of RBC. They also require availability of larger volume of patient sample, as plasma is difficult to separate cleanly from very small quantity of samples of blood.
The above-mentioned factors make the conventional methods unacceptable for ‘point of care’ (POC) or ‘near patient’ applications, which involve rapid test results with user-friendly equipments used by minimally trained staff. Increasingly, the need is felt for ‘Point of Care’ devices, which can help deliver quantitative results of similar quality as obtained from laboratory analyzers.
Several patents describe materials and devices to accomplish RBC and particulate removal from biological fluids. U.S. Pat. Nos. 4,816,224, 5,186,843, 5,240,862 teach variety of materials and devices which can be used to separate RBC from whole blood. U.S. Pat. Nos. 4,980,297, 5,135,719, 5,064,541, 5,139,685, EP1315553, EP1096254B1, U.S. Pat. Nos. 6,296,126B1, 6,197,598B1 and 6,391,265B1, 7,279,136 describe devices and methods for separating plasma from whole blood and integrating these devices with various detection methods and ‘Point of Care’ instruments for detection and quantitation of analytes.
Many of the above inventions suffer from the fact that the fluid path for sample fluid is relatively short in the transverse direction resulting in low efficiencies of retention and leakage of particulate matter. This problem is overcome in some prior art inventions by adopting lateral flow of the sample which results in chromatographic separation of particulate matter and plasma is separated. Further retention is sought to be improved by region specific compression.
U.S. Pat. No. 6,391,265 titled “Devices incorporating filters for filtering fluid samples” discloses devices and use thereof, the device comprising a filter and a means for region specific compression of the filter. Alternatively, a single step assay device whereby fluid movement through the device occurs substantially solely due to action of capillary force, the device comprising a filter, a region containing the filter, a fluid access port to the region containing the filter, a means for retarding movement of particles through a peripheral filter surface, a fluid egress port from the region containing the filter, a lateral fluid flow path through the filter connecting the fluid access port to the fluid egress port, whereby sample fluid substantially devoid of particulate matter is released from the filter through the egress port; and, an exit region fluidly connected to the egress port.
The above mentioned prior art devices which do utilize long path length by lateral flow of sample and selective compression of filter and sealing of extraneous capillaries which can cause leakage of particulate matter tend to use large amounts of blood to achieve very small amounts of plasma. The plasma is obtained in a reaction zone capillary of less than 25 micron channel height.
U.S. Pat. No. 5,064,541 shows the uses of agglutinating agents in the sintered porous material to retain RBC and remove the separated plasma by wicking it with a matrix. By making the matrix of reproducible fluid uptake a measured amount of plasma is available in the matrix.
Due to short path lengths, not only do these devices suffer from poor retention but the free or pure plasma is not available as there is no provision to remove the pure plasma from the matrix.
US Patent Application 2004/0035792A1 describe a method where whole blood is placed into a feed chamber which is isolated by means of a membrane from a closed cavity having small height such that its capillary forces are higher than that of the separation membrane. The whole blood is filtered by means of forces of suction, pressure, capillary forces and or hydrostatic pressure to the capillary with small height where filtered plasma in pure liquid form is available. A method of removal of pure plasma by application of pressure on the sample (blood) feed or by applying suction on the plasma collected in the small height capillary is also described.
It is well known that when whole blood is filtered by applying pressure, it has to be done extremely slowly as the interstecial space of filter are filled with the RBC and with increasing pressure they will rupture contaminating the plasma. This makes application of pressure to recover plasma impractical in above invention. Removal of plasma by vacuum and making it available to reaction zones with large capillary heights or cuvettes or tubes requires complicated mechanical device and driving systems making the device costly and more difficult to use.
U.S. Pat. No. 6,296,126B/describes micromachined device with wedge shaped cutouts in contact with separated plasma in the matrix. The wedge shaped cutouts facilitate the removal of liquid from the matrix.
These devices suffer from very low recoveries of pure liquid and high cost of manufacture.
U.S. Pat. No. 7,404,931 discloses a device for plasma separation which has a separation element designed to comprise a separation zone and a transport zone. The separation zone separates the plasma from whole blood and transports it to the transport zone. A plunger system is incorporated in the device to detach the transport zone from the separation zone to collect the separated plasma.
The device of the '931 patent is restricted in its application in the sense that it completely detaches the transport zone from the separation zone thus negating any chance of recovery of residual plasma from the separation zone. In case of limited availability of blood sample, particularly with neonatal blood, such recovery of residual plasma become a critical parameter of checking the efficiency of such devices.
For ‘Point of Care’ (POC) tests with whole blood, it is very desirable to work with minimum amount of blood and obtain a quantitative amount of pure, undiluted plasma for analysis. With the advent of microfluidic devices it is even more desirable to have a means to deliver required amount of plasma to capillaries comprising reaction and reading zones of the POC devices.
The problem with above known methods to deliver plasma to capillaries is even more acute when the capillary itself is the reaction and reading zone. To achieve high sensitivities it is often necessary to keep capillary height of reading zones to greater than 25 microns and more close to 100 microns or greater. In such capillaries the filtered fluid accumulated at the distal end of the filter remains entrapped in the matrix and does not migrate into large height capillaries or reaction zones or reservoir from where it may be transferred to reaction cuvettes as the capillary forces are not strong enough to draw the filtered fluid out of the matrix.
Thus there exists a need for a simple, cost effective device which allows rapid and efficient removal of particulate matter from fluids to be analysed and delivers the filtered fluid in free form or droplet form without entrapment in the matrix or small height capillary so that the fluid can flow into open tanks or reaction zones or other zones with capillary forces lower than that of the matrix or capillary holding the filtered fluid. Moreover, such a device should be able to effectively collect a substantial volume of residual plasma that has failed to be transported to a transport zone in a first filter pass.
Such a device may be used separately or it can be easily integrated for qualitative and quantitative analysis with many different types of Point of Care and other instruments.